Invasive detection of colonic biomarkers

ABSTRACT

A noninvasive method utilizing feces, containing sloughed colonocytes, as a sensitive technique for detecting diagnostic colonic biomarkers as well as a method for isolating poly A+ RNA from feces. The method allows the isolation and quantitation of specific eukarotic mRNAs as candidate biomarkers for colon cancer isolated from feces.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Continuation-in-part of PCT Application No. PCT/US98/06698 filedApr. 3, 1998, which claims priority of U.S. Provisional PatentApplication Serial No. 60/043,048, filed Apr. 4, 1997, both beingincorporated herein by reference, is hereby claimed.

REFERENCE TO A “MICROFICHE APPENDIX”

[0002] Not applicable

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] Not applicable

BACKGROUND OF THE INVENTION

[0004] 1. Field of the Invention

[0005] The present invention relates to methods for the noninvasivedetection of colonic biomarkers using fecal messenger RNA (mRNA). Moreparticularly, the present invention relates to methods for the isolationof poly A+ RNA from feces, and includes the subsequent detection of, andquantitation of, particular mRNAs that correlate with a patient'sdiagnosis and/or prognosis of colon cancer thereby providing methods fornoninvasively diagnosing and/or prognosticating colon cancer in apatient. One embodiment of the present invention relates to thedetection of, and quantitation of, mRNA from sloughed colon cells infeces encoding particular isozymes of protein kinase C (PKC) whoselevels are correlative with and predictive of colon cancer in a patient.Methods including semi-quantitative RT-PCR and biochip microarraytechnology may be made to assay and evaluate the fecal poly A+ RNA.

[0006] 2. General Background

[0007] Since colon cancer is the second most common cause of U.S. cancerdeaths and since early detection can result in a high cure rate, anaccurate screening method for colon cancer is imperative. Currentdetection methods have many drawbacks. For example, fecal occult bloodscreening can produce false positive results due to meat consumption,iron supplement intake and other common behaviors. The other routinescreening technique, sigmoidoscopy, is an invasive expensive procedurewhich has inherent risks of perforation, reaction to sedative, orbleeding. In addition, the efficacy of sigmoidoscopy screening remainsunproven (Levin, 1996). Because of these limitations, colon cancer curerates have not improved in the past 30 year (Silverberg, 1988, WFR/AICR,1997). Therefore, an accurate technique to detect early changesassociated with the tumorigenic process is imperative in order todecrease the mortality from colon cancer.

[0008] Screening of colorectal cancer is recommended for all personsaged 50 and older with annual fecal occult blood testing orsigmoidoscopy, or both (Levin, 1996). However, each of these tests haslimitations related to sensitivity and specificity (Levin, 1996). Thepresence of colorectal and pancreatic tumors has been detected in thestool and colonic effluent of patients by noninvasive methods based onthe molecular pathogenesis of the disease (Sidransky, 1992: Tobi, 1994;Caldas, 1994). These protocols utilize DNA extraction procedures and thedetection of oncogene mutations using PCR. The major disadvantage ofthis methodology is that it will not detect alterations in geneexpression. Our methodology can quantitate the expression of anyrelevant gene by isolating and amplifying mRNA derived from fecalmaterial containing sloughed colonocytes.

[0009] A sensitive molecular technique for the detection of colon canceris important since early diagnosis can substantially reduce mortality(Levin, 1996). Our method is noninvasive, highly sensitive and specific.Our protocol is unique because it will determine colonic expression ofany gene (e.g., tumor suppressor gene, oncogene), and provides earlysensitive prognostic information and greatly enhances current methods ofdietary and pharmacologic risk assessment.

SUMMARY OF THE PRESENT INVENTION

[0010] The present invention relates to a novel non-invasive technologyto detect changes in colonocyte gene expression associated with earlystages of colon tumorigenesis. This invention also covers the firstknown methods to isolate poly A+ RNA from feces. This methodology hasthe advantage of utilizing a fecal sample, which contains sloughed coloncells. Therefore, it does not require anesthesia or cause any discomfortto the patient. In addition, the invention utilizes a novel mRNAisolation process that results in an unexpectedly high yield andstability of isolated fecal mRNA, and utilizes an exquisitely sensitivetechnique, rapid competitive polymerase chain reaction (Jiang, 1996),developed by the inventors, to detect and quantify mRNA markers of thetumorigenic process. Thousands of gene markers for the tumorigenicprocess are assayable in the practice of the present invention. Thesemarkers include, but are not limited to, PKC isozymes such as, forexample, PKC βII (PKC beta II) and PKC ζ (PKC zeta), where, for example,levels of these particular isozymes in feces are correlative of andpredictive of the presence of, and development of colon cancer in a ratcolon cancer model (Davidson, 1998). We have also successfully isolatedpoly A+ RNA from rectal vault eluate isolated at the initiation ofcolonoscopy. Yields from fecal eluate are generally in the range of0.3-1.5 μg poly A+ RNA isolated per subject.

[0011] The pathogenesis of colon cancer is a multi-step process, inwhich tumor suppressor genes, oncogenes and other molecules involved insignal transduction are affected (Fearon, 1997). It is now clear thesignals mediated via select isozymes of protein kinase C (PKC) areinvolved in colonic tumor development (Sakanoue et al., 1991; Kopp etal. 1991; Baum et al., 1990). PKCs are a family of serine-threoninekinases thought to regulate colonic cell proliferation, differentiationand programmed cell death. PKCs can be divided into three differentsub-categories based on the cofactors needed for activation: classicalPKCs (α, βI, βII and γ) require diacylglycerol (DAG) and Ca²⁺ foractivation; novel PKCs (δ, θ, η and ε) are Ca²⁺ independent, butactivated by DAG; and atypical PKCs (λ, ι and ζ) are Ca²⁺ and DAGindependent. Although these isozymes are enzymatically similar, in vivo,they have different expression patterns depending on tissue and celltype (Blobe et al., 1996).

[0012] PKC βII protein is generally found in very low levels in normalrat colonic mucosa (Davidson et al., 1994). However, βII protein levelsincrease in colonic tumors as compared with normal colonic mucosa(Craven et al., 1992; Wali et al., 1995). In contrast, PKC ζ mRNA levelsare significantly lower in human colorectal tumors than in normalcolonic mucosa (Kuranami et al., 1995). PKC ζ protein levels also aresignificantly lower in preneoplastic colonic epithelium from ratsinjected with azoxymethane (AOM) as compared with saline-injectedcontrol rats (Wali et al., 1995; Roy et al., 1995; Jiang et al., 1997).Therefore PKC βII and ζ may serve as biomarkers to monitor thedevelopment of colon cancer.

[0013] In summary, no one has reported the isolation of intact poly A+RNA from fecal material or rectal eluates obtained at colonoscopy.Utilization of this noninvasive procedure combined with either RT-PCRanalyses or genechip microarrays is novel.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] For a further understanding of the nature and objects of thepresent invention, reference should be had to the following detaileddescription taken in conjunction with the accompanying drawings, inwhich like parts are given like reference numerals and wherein:

[0015]FIG. 1 shows representative competitive PCR products fordetermination of Liver-Fatty Acid Binding Protein (L-FABP) expression infecal poly A+ RNA. Lane 1, marker; lane 2, rat colonic mucosa(standard); lanes 3-6, rat poly A+ RNA. Upper band is amplified sampleband (390 bp); lower band is amplified internal standard (336 bp).

[0016]FIG. 2 shows a representative gel of RC-PCR products of PKC βII.Lane 1, marker; lanes 2-5 fecal poly A+ samples. Upper band is theamplified sample band (419 bp); the lower band is the amplified internalstandard (361 bp).

[0017]FIG. 3 shows a representative gel of RT-PCR products of PKC βI andPKC γ in brain but not in fecal poly A++RNA. Lane 1, marker; lane 2, PKCβI in brain (639 bp); lanes 3 and 4, PKC βI in fecal poly A+ RNA; lane5, PKC γ in brain (347 bp); lanes 6 and 7, PKC γ in fecal poly A+ RNA.

[0018]FIG. 4 shows expression of PKC βII in fecal poly A+ RNA or colonicmucosal RNA. Rats were injected with azoxymethane (AOM) or saline twice.Feces were collected 36 weeks after the second injection and poly A+ RNAwas isolated. Colonic mucosa was scraped and total RNA was isolated.Quantitative RC-PCR was performed using primers specific for PKC βII.PCR products were separated on 4% agarose gels, stained with ethidiumbromide, photographed and scanned on a densitometer to quantitate.Y-axis represents band intensities (OD×mm²). (A) Expression of PKC βIIin fecal poly A+ RNA from rats with or without tumors (mean±SEM;P=0.026; n=12-29). (B) Expression of PKC βII in colonic mucosal RNA fromrats injected with AOM or saline (mean±SEM; P=0.036; n=16-20). “BI” is“band intensity”, “T” is “tumor”, “NT” is “no tumor”, “I” is“injection”, and “S” is “saline”.

[0019]FIG. 5 shows expression of PKC ζ in fecal poly A+ RNA from ratsinjected with AOM or saline. See FIG. 4 legend for further details(mean±SEM: P=0.017; n=21-22).

[0020]FIG. 6 shows expression of PKC βII/PKC ζ ratio in fecal poly A+RNA from rats with or without tumors. See FIG. 4 legend for furtherdetails (mean±SEM; P=0.025; n=9-26).

[0021]FIG. 7 shows a representative agarose gel of PKC βII (A) and PKC ζ(B) RT-PCR products from human rectal vault eluate obtained at theinitiation of colonoscopy and from freshly isolated human fecal poly A+RNA. Lanes 1 and 2, amplification from rectal vault eluate poly A+ RNA;lane 3, amplification from fecal poly A+ RNA; lane 4, minus RT negativecontrol; lane 5, amplification from human brain poly A+ RNA (positivecontrol); lane 6, base pair marker. PKC βII product is 280 bp, PKC ζproduct is 216 bp.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0022] The development of noninvasive techniques, as shown in thisinvention, provides early sensitive prognostic information and willgreatly enhance the current methods of dietary, pharmacologic, andcancer risk assessment. The present invention describes a noninvasivemethod utilizing feces containing sloughed colonocytes as a sensitivetechnique for detecting diagnostic biomarkers in the colon. Byincorporating a novel method of isolating fecal mRNA and by utilizingthe exquisite sensitivity of quantitative rapid competitive reversetranscriptase polymerase chain reaction (RC-PCR), the method is capableof isolating and quantitating specific messenger RNAs (mRNAs) ascandidate biomarkers in feces. The RNA can also be assayed and evaluatedusing nucleic acid “biochip”/“microarray” technology as described belowand as understood in the art. This technology allows for large-scalehigh-throughput monitoring of gene expression patterns of up to 40,000genes (Lipshutz, 1999). Further, the present invention has recognized acorrelation between levels of particular biomarkers and the presence ofand development of colon cancer.

[0023] For example, but not in a limiting sense, the present inventionrecognizes that PKC βII expression in fecal poly A+ RNA is positivelycorrelated with tumor incidence and the expression of PKC ζ isnegatively correlated with tumor incidence (Davidson, 1998).

[0024] The method of the present invention involves a novel technique ofisolating mRNA from feces that results in, inter alia, substantialimprovement in yield, and stability of isolated poly A+ RNA fromexfoliated colonocytes in feces, in a substantially reduced amount oftime compared with the only other known techniques in the art (thetechnique of Davidson, 1995).

[0025] Approximately one-sixth to one-third of normal adult colonicepithelial cells are shed daily (Potten, 1979). Isolation of colonocytesfrom feces has been reported by another group (Albaugh, 1992). Thismethod is very time consuming and results in an extremely low yield suchthat useful diagnostic tests on the isolated cells are limited and verylabor intensive. We therefore designed a technique to directly isolatepoly A+ RNA from feces containing exfoliated colonocytes. The poly A+RNA isolated can be used to probe for early markers for colon cancer orother colorectal diseases.

[0026] Specifically, we have redesigned the protocol of the prior art(for example, Davidson, 1995) to significantly simplify and enhance theprocess, resulting in a greatly enhanced yield. In addition, we havecombined the improved isolation protocol with an extremely sensitivedetection technique, called rapid competitive polymerase chain reaction(RC-PCR), a technology developed in our laboratory.

[0027] The original method (Davidson, 1995) involved the isolation oftotal RNA from feces followed by poly A+ RNA isolation, which couldsubsequently be utilized for assessment of colon cancer biomarkers. Thisolder methodology resulted in a relatively low yield of poly A+ RNA,thereby limiting the diagnostic tests which could be performed. Themodifications, detailed below, result in approximately 10-fold increasein poly A+ RNA yield, allowing for extensive screening of various coloncancer biomarkers. In addition, the method is straight-forward and couldbe performed by a trained technician. Several samples (up to 12 or more)can be processed at once.

[0028] The refined RNA isolation technology of the present invention hasbeen validated using the rat chemical carcinogen model. Specifically, wehave demonstrated that protein kinase C (PKC) βII and PKC ζ inexfoliated colonocytes may serve as noninvasive markers for developmentof colon tumors (Davidson, 1998).

[0029] The improved method is an improvement on the basic method setforth by Laurie A. Davidson, Yi-Hai Jiang, Joanne R. Lupton, and RobertS. Chapkin in Noninvasive Detection of Putative Biomarkers for ColonCancer Using Fecal Messenger RNA, published in Cancer Epidemiology,Biomarkers & Prevention, Vol. 4, 643-647, September, 1995—this paper ishereby incorporated by reference in its entirety. Instant improvementsinclude, for example, poly A+ RNA is directly isolated from feces usingoligo dT cellulose based methodology. The previous published report(Davidson, 1995) involved total RNA isolation from feces followed bypoly A+ isolation from the total RNA preparation. The improved methodshortens the poly A+ RNA isolation to 5 h (from 2 d with the previousmethodology) and significantly increases yield by 5-10 fold.

[0030] In still another feature of the present invention, and animprovement over the prior art, the present invention is suitable forthe detection, and quantitation of specific biomarkers whose expressionin colon cells and thus, in poly A+ RNA isolated from feces, correlateswith and is predictive of states of colon cancer in a patient.

[0031] For example, the present invention shows that PKC βII and PKC ζare suitable as biomarkers for monitoring the development of coloncancer. The modulation of these putative biomarkers—affected by thepresence or absence of colon tumors is shown herein. Weanling rats wereinjected with saline (control) or carcinogen (azoxymethane). Fresh fecalsamples (n=6 per diet) were collected 36 weeks post injection, poly A+RNA was isolated and quantitative RC-PCR performed using primers to PKCβII and ζ. PKC isozyme expression was altered by the presence of tumors(P<0.05), with tumor bearing animals having a 3-fold higher βIIexpression and 6-fold lower ζ expression in exfoliated colonocytes thannon-tumor bearing animals. We propose that expression of PKC βII and ζin exfoliated colonocytes may serve as a noninvasive marker fordevelopment of colon tumors.

[0032] Also novel is the use of the rapid competitive PCR method (asfirst disclosed in Jiang, 1996) to sensitively quantify biomarkerexpression in fecal poly A+ RNA. This method is described in detail inRapid competitive PCR determination of relative gene expression inlimiting tissue samples, Yi-Hai Jiang, Laurie A. Davidson, Joanne R.Lupton, and Robert S. Chapkin, Clinical Chemistry, 42:2, 227-231 (1996),which is hereby incorporated by reference in its entirety. This methodis ideal for limited RNA samples, since it requires only a single PCRreaction in order to determine relative gene expression. In contrast,the more traditional mimic reverse transcriptase (RT)-PCR techniquerequires a series of 5 to 7 PCR reactions in order to quantitate geneexpression.

[0033] For example and for illustrative purposes only, at least thefollowing features of the present invention are novel over the priorart: (1) Direct isolation of poly A+ RNA from feces or rectal eluates;(2) Ten-fold increase in poly A+ yield with decrease in processing timeby more than 50%; (3) Identification of protein kinase C (PKC) βII as amarker for colon cancer; (5) Use of the novel relative competitiveRC-PCR method to detect and quantify markers of colon cancer in fecescontaining exfoliated colon cells; and (6) Validation of fecalhomogenate stability after processing and storage prior to poly A+isolation.

[0034] The methods of the present invention can be utilized to detectpredictive risk markers for colon cancer including, but not limited to,biomarkers such as:

[0035] Acyl CoA Binding Protein (ACBP) expression

[0036] Arginase expression

[0037] bax expression

[0038] bcl-2 expression

[0039] Bcl-XL expression

[0040] Bcl-Xs expression

[0041] c-myc expression

[0042] Carcinoembryonic Antigen (CEA) and Nonspecific CrossreactingAntigen (NCA) expression

[0043] CD44 Glycoprotein expression

[0044] Cyclin-dependent kinase inhibitors (p27, p16ink4) expression

[0045] Cyclin-dependent kinase cdk2/cdc2, cyclin D1, and cdk4 expression

[0046] Cyclooxygenase I and II

[0047] Decay Activating Factor expression

[0048] E-Cadherin cell adhesion molecule expression

[0049] Epidermal Growth Factor Receptor (EGFR) expression

[0050] Fatty Acid Synthase expression

[0051] Fecal alpha-1 Antitrypsin expression

[0052] GDP-L-fucose:beta-D-galactoside-alpha-2-L-fucosyltranferaseexpression

[0053] Gluthathione S-Transferase expression

[0054] Histone H3 expression

[0055] Interleukin 1 and 2 expression

[0056] Liver and Intestinal Fatty Acid Binding Protein expression

[0057] hTERT expression

[0058] Mitogen-activated protein kinase (MAP kinase) expression

[0059] MAP kinase phosphatase-1 expression

[0060] NO synthase, inducible expression

[0061] Ornithine Decarboxylase expression

[0062] p21 waf 1/cip 1 expression

[0063] P-glycoprotein, the mdr gene product expression

[0064] Plasminogen Activator expression

[0065] Proliferating cell nuclear antigen (PCNA) expression

[0066] Prostaglandin Synthase Type II (COX II) expression

[0067] Protein Kinase A, Type I and II Isozyme expression

[0068] Protein Kinase C α, βII, δ, ε, λ, ι, μ, ζ expression

[0069] Ras oncogene expression

[0070] Ras oncogene mutations

[0071] Stearoyl-CoA desaturase expression

[0072] Sterol Carrier Protein-2 (SCP-2) expression

[0073] Telomerase expression

[0074] Transforming Growth Factor-beta I and II expression

[0075] Transforming Growth Factor-beta type II Receptor expression andmutations

[0076] Tumor Necrosis Factor Alpha expression

[0077] Tumor suppressor gene APC mutations

[0078] Tumor suppressor gene p53 mutations and expression

[0079] Tumor suppressor gene retinoblastoma (Rb) protein expression

[0080] Villin expression

[0081] 1,25-dihydroxyvitamin D3 Receptor expression, and

[0082] 13-hydroxyoctadecadienoic acid (13-HODE) dehydrogenaseexpression.

[0083] The present invention is suitable for noninvasive detection ofany diagnostic gene or panel of genes including PKC isozymes aspredictive risk markers for human colon cancer. We have alreadyvalidated the use of select PKC isozymes as predictive risk markersusing the rat experimental colon cancer model (Davidson, 1998). Inaddition, we have isolated human poly A+ RNA from feces and rectaleluates and detected the presence of PKC isozymes.

[0084] Additionally, the present invention, using, for example the ratcolon cancer model, relates to the determination of the temporal effectsof carcinogen on select PKC isozyme fecal mRNAs.

[0085] The development of noninvasive techniques, as shown in thisinvention, provide early sensitive prognostic information and greatlyenhance current methods of dietary and pharmacologic risk assessment.The method reported herein is novel since it is the first to report thatpoly A+ RNA from exfoliated colonocytes can be isolated directly fromfeces or rectal eluates and can be used to probe for markers of coloncancer. Several markers have also been identified that are present infecal poly A+ RNA that predict for colon cancer.

EXPERIMENT 1 Utilization of Isolated Fecal Poly A+ RNA to Detect ColonCancer Markers

[0086] Further details related to this method may be found in thearticle by Laurie A. Davidson, Christin M. Aymond, Yi-Hai Jiang, NancyD. Turner, Joanne R. Lupton and Robert S. Chapkin, entitled“Non-invasive detection of fecal protein kinase C βII and ζ messengerRNA: putative biomarkers for colon cancer”, published in Carcinogenesis,vol. 19, no. 2, pp. 253-257, 1998, which is hereby incorporated byreference in its entirety.

Experimental Methods

[0087] Isolation of poly A+RNA from feces:

[0088] 1. Collect 0.3-2.0 g of rat or human feces. Within 30 min ofdefecation, add 10 volumes of Lysis Solution (available from Poly A+Pure Kit, Ambion, Austin, Tex.) (“Ambion Kit”). Homogenize feces with apestle. This homogenate can be stored at −80° C. for several monthsbefore further processing.

[0089] 2. Transfer homogenate to sterile 50 ml conical Falcon tube andmeasure the volume. Add 2 vol Dilution Buffer (Ambion Kit). Mix byinversion for 10 sec. Centrifuge at 4,000×g, 15 min, 4° C. Transfersupernatant to a new sterile 50 ml Falcon tube.

[0090] 3. Add oligo dT cellulose (Ambion Kit), an amount equal to 10% ofthe starting fecal weight. Mix by inversion to resuspend the oligo dTresin.

[0091] 4. Rock the tube on a horizontal shaker at 100-150 rpm at roomtemperature for 1 hr.

[0092] 5. Pellet the oligo dT resin by centrifuging at 4,000×g, 3 min,4° C. Remove and discard the supernatant.

[0093] 6. Resuspend the resin with 6-10 ml Binding Buffer (Ambion Kit)and mix well. Pellet resin as described in step 5 and discard. Repeatthis two more times. 7. Resuspend resin with 6-10 ml Wash Buffer (AmbionKit) and mix well. Centrifuge as described in step 5 and discardsupernatant. Repeat this wash two more times.

[0094] 8. Resuspend the resin in 1-2 ml wash buffer and transfer to aspin column in a 1.5 ml microfuge tube (Ambion Kit). Centrifuge at5,000×g, 10 sec, room temperature to remove the supernatant. Place spincolumn into a new microfuge tube.

[0095] 9. Add 300 μl Elution Buffer (Ambion Kit) which has beenpre-warmed to 65° C. Immediately centrifuge at 5,000×g, roomtemperature, 30 sec and save the eluate. Add another 300 μl pre-warmedElution Buffer and centrifuge at 5,000×g, room temperature, 30 sec.Combine eluate with previous eluate. Discard the spin column.

[0096] 10. Precipitate the poly A+ RNA by adding 60 μl 5M ammoniumacetate, 10 μg glycogen and 2.5 vol 100% ethanol. Place at −80° C. for 1h. Recover poly A+ RNA by centrifugation at 12,000×g, 20 min, 4° C.Remove and discard supernatant, add 0.5 ml chilled 80% ethanol to thetube, invert tube gently. Resuspend the poly A+ pellet in 60-200 μlwater/0.1 mM EDTA. Vortex gently to resuspend.

[0097] This purified A+ RNA is used for colon cancer biomarker studiessuch as those detailed below:

Results

[0098] Using the method described above, fecal poly A+ RNA from ratsinjected with carcinogen or saline (control) was examined for coloncancer biomarkers. We determined that protein kinase C βII expression infecal poly A+ RNA is positively correlated with colon tumor incidence(FIG. 4A), while protein kinase C ζ is negatively correlated with tumorincidence (FIG. 5).

[0099] The ratio of PKC βII to ζ is also strongly correlated with tumorpresence (Table 1 and FIG. 6). TABLE 1 Relationship between PKC βII:ζratio and tumor incidence. PKC βII:ζ ratio Animals with tumors 4.27 ±2.37 p = 0.02 Animals without tumors 0.71 ± 0.14

EXPERIMENT 2 Utilization of Isolated Fecal Poly A+ RNA to Detect ColonCancer Markers II

[0100] Liver fatty acid binding protein (L-FABP) and intestinal fattyacid binding protein (I-FABP), expressed in colonocytes, are additionalcolon cancer biomarkers. Data indicates that expression of L-FABP andI-FABP are significantly depressed in carcinogen treated animals. FIG. 2documents a typical gel containing rapid competitive PCR products forL-FABP. The upper band represents the sample (390 base pairs), whereasthe lower band is the internal standard (336 base pairs).

EXPERIMENT 3 Human Clinical Trials Methodology

[0101] Clinical.

[0102] Patients presenting for colonoscopy are individually typed as: 1)being free of colon cancer, 2) having adenomatous polyps (consideredpreneoplastic), or 3) having colon cancer (presenting histologicalevidence of adenocarcinomas). Thirty subjects for each group arerecruited in order to reduce the effect of individual variation on theanalysis. The sample size is based upon testing equality of means with aα=0.05 and detecting a difference of size σ with a probability of 95%(Pearson and Hartley, 1966). To achieve this level of statistical powerrequires 26 individuals. Thus 30 patients protect against loss of powerif a sample becomes damaged during storage or analysis. Because patientsrandomly present for treatment, and the disease state will not be acontrolled factor, we assume the data will be randomly distributed amongthe potential population. Because patients with cancer are the limitingfactor in sample collection, the first 30 individuals with cancer arethose selected for inclusion in the study. In order to adjust forvariation related to patient age, individuals free of colon cancer andthose with polyps are age-matched to patients with colon cancer.Further, patients with polyps or free of pathology are selected after asample is collected from a cancer patient.

[0103] A patient will follow a bowel preparation schedule prior tocolonoscopy. Patients will receive the Golytely™ (3-4 L, Braintree Labs,Braintree, Mass.) colonoscopic preparation. This preparation wasselected because it preserves surface epithelial and goblet cells andhas minimal effects on a variety of colon cancer risk biomarkers. At thetime of colonoscopy, the rectal vault eluate (5-50 ml) will be suctionedthrough the scope into a disposable suction trap. The trap will beremoved and its contents transferred immediately into Lysis solution(from poly A+ Pure Kit, Ambion, Austin, Tex.) and placed on ice untilthe end of the case (<45 min). Samples will subsequently be stored at−80° C. until transported to the analysis lab for further processing.

[0104] Laboratory.

[0105] Samples are stored at −80° C. until being thawed on ice, and thehomogenate transferred to sterile tubes and the volume measured.Dilution buffer (Ambion Poly A+ Pure Kit) is added and the contentsmixed by inversion and then centrifuged at 4,000×g for 15 min at 4° C.Oligo dT resin is added to the sample and the supernatant is then mixedby inversion to resuspend the oligo dT resin prior to rocking the tubeon a horizontal shaker. Following centrifugation, the resin pellet isresuspended with binding buffer (Ambion Kit). The resin is thenpelleted, supernatant discarded and resulting poly A+ RNA eluted fromthe resin and used to determine biomarker prevalence (Davidson et al.,1995). The biomarkers chosen for analysis are PKC βII and PKC ζ, basedon our previous research indicating the βII isoform is positivelycorrelated with colon tumor incidence (FIG. 4A), and the ζ isoform isnegatively correlated with tumor incidence. In addition, cyclin D₁(Arber, 1996), survivin (Lacasse, 1998), cyclooxygenase type II(Kutchera, 1996), p53 (El-Mahdani, 1997), and human telomerase reversetranscriptase (hTERT) (Sumida, 1999) were selected based on the citedresearch indicating a strong correlation between mRNA expression andtumor incidence. RC-PCR (Jiang et al., 1996) is used to detect the levelof expression for each of the biomarkers.

[0106] A representative agarose gel showing quantitative RT-PCR of humanPKC βII and ζ is shown in FIG. 7. The fidelity of all PCR reactions wasconfirmed by DNA sequencing (Davidson, 1994). Negative controlsprocessed without RT yielded no detectable amplified products indicatingthe absence of DNA contamination. Comparable results were obtained fromfreshly isolated fecal samples (refer to FIG. 7 for details).

[0107] Data is analyzed using the GLM models of SAS. Differences betweengroups are determined by orthogonal contrasts. Data from healthyindividuals are compared with those having either polyps or cancer todetermine if the presence of the pathologies affect the relative mRNAexpression for the genes with biomarker potential. In addition, acontrast of the individuals with polyps vs those with cancer isperformed to determine if the expression changes with stage of thetumorigenic process.

EXPERIMENT 4 Detection of Fecal Protein Kinase C βII and ζ Messenger RNAColon Cancer Biomarkers

[0108] The animal use protocol conformed to NIH guidelines and wasapproved by the University Animal Care Committee of Texas A&MUniversity. Forty-eight male weanling Sprague-Dawley rats (HarlanSprague-Dawley, Houston, Tex.) were randomly divided into two groups aspreviously described (Chang et al., 1997) and given two types ofinjection (carcinogen or saline). Animals were housed individually insuspended cages in a temperature and humidity controlled animal facilitywith a 12 h light/dark cycle. Food and distilled water were freelyavailable. Forty-eight h food intakes and fecal outputs were measuredduring the study. Body weights were recorded weekly.

[0109] Carcinogen Administration and Fecal Collection:

[0110] After a 2 week acclimation period, rats were given two s.c.injection of AOM (Sigma Chemical Co., St. Louis, Mo.) at a dose of 15mg/kg body weight or an equal volume of saline (one injection/week)(Chang et al., 1997). Animals were killed by CO₂ asphyxiation 36 weeksafter the second injection. The colon was subsequently removed and themost distal fecal pellet collected. The pellet was immediately placed inLysis solution for RNA isolation (Ambion Totally RNA kit, Austin, Tex.).The colon was then visually inspected for tumors and tumor typing wasdetermined (Chang et al., 1997). Briefly, tissue sections were fixed in4% buffered formalin, embedded in paraffin, and stained with eosin andhematoxylin. Slides were then microscopically evaluated for tumors aspreviously described (Chang et al., 1997). Following removal ofsuspected tumors for histological evaluation, the remaining colonicsections were gently scraped with a microscopic slide and the mucosaused for determination of steady-state levels of PKC isozyme mRNA.Histological evaluation of this method indicated that epithelial cellsand lamina propria down to the muscularis mucosa were removed (Lee etal., 1992).

[0111] RNA Isolation:

[0112] Fecal poly A+ RNA was prepared as described above. Quantificationof fecal poly A+ RNA was performed as previously described (Davidson etal., 1995). Briefly, samples were quantitated by blotting fecal poly A+RNA onto a positively charged nylon membrane (Roche, Indianapolis,Ind.). A biotinylated oligo (dT) probe (Promega, Madison, Wis.) washybridized to the poly A+ RNA followed by detection withstreptavidin-alkaline phosphatase. Dilutions of colonic musocal totalRNA of known concentration (as determined from absorbance at 260 nm)were also blotted to generate a standard curve. For concentrationcalculations, it was assumed that poly A+ RNA constitutes 3% of totalRNA.

[0113] Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Assayfor Negative Controls (PKC γ and PKC βI)

[0114] Aliquots of 40 ng fecal poly A+ RNA in a 50 μl reaction werereverse transcribed to generate first strand cDNA using Superscript IIreverse transcriptase (Gibco-BRL, Gaithersburg, Md.) as previouslydescribed (Davidson et al., 1995). PCR was performed using Expand HighFidelity DNA polymerase (Roche, Indianapolis, IN). The 50 μl PCRreaction consisted of 1×PCR buffer, 2% DMSO, 0.05 mM dNTPs, 1.5 mMMgCl₂, 20 pmol each of forward and reverse primer, 2.6U Expand HighFidelity DNA polymerase and 10 μl of RT reaction. Rat brain cDNA was runas a positive control. PCR was performed using a Perkin-Elmer 2400thermal cycler (Perkin-Elmer, Foster City, Calif.) with the followingamplification program: 15 s denaturation (94°), 15 s annealing (59° C.)and 45 s extension (74° C.) for 40 cycles. PCR products were analyzed ona 4% agarose gel followed by ethidium bromide staining. All PCR productswere sequenced to ensure the fidelity of amplification (Davidson et al.,1994). The primer pair for PKC γ was as follows (347 bp); forward,5′-TTGATGGGGAAGATGAGGAGG-3′, Sequence ID No. 1; reverse,5′-GAAATCAGCTTGGTCGATGCTG-3′, Sequence ID No. 2. The primer pair for PKCβI was as follows (639 (bp): forward, 5′-TGTGATGGAGTATGTGAACGGGGG-3′,Sequence ID No. 3; reverse, 5′-TCGAAGTTGGAGGTGTCTCGCTTG-3′, Sequence IDNo. 4.

[0115] Rapid Competitive Reverse Transcription-Polvmerase Chain ReactionAssay for Fecal and Mucosal PKC ζ and βII

[0116] Rapid competitive RT-PCR was performed in order tosemi-quantitatively determine the PKC ζ and βII fecal and mucosal mRNAlevels as previously described (Jiang et al., 1996). Using this method,relative gene expression was determined by co-amplifying an exogenousDNA target (‘internal standard’) with a different size than the samplecDNA but with identical 5′ and 3′ ends. This allows for competitionbetween the sample cDNA and the internal standards for primers (Jiang etal., 1996). Internal standards were prepared as described previously(Davidson et al., 1995). Fecal poly A+ RNA was processed as describedabove. In addition, 6 μg of mucosal total RNA was reverse transcribed ina 50 μl reaction and 10 μl was amplified in the presence of either 140fg of PKC ζ internal standard or 31.2 fg PKC βII internal standard. Theprimer pair for the PKC ζ internal standard was (561 bp): forward,5′-CGATGGGGTGGATGGGATCAAAA-3′, Sequence ID No. 5; reverse,5′-GTATTCATGTCAGGGTTGTCTGGATTTCGGGGGCG-3′, Sequence ID No. 6, and forPKC ζ was (680 bp): forward, 5′-CGATGGGGTGGATGGGATCAAAA-3′, Sequence IDNo. 7; reverse, 5′-GTATTCATGTCAGGGTTGTCTG-3′, Sequence ID No. 8. Theprimer pair for PKC βII internal standard was (361 bp): forward,5′-TATCTGGGATGGGGTGACAACCGAGATCATTGCTTA-3′, Sequence ID No. 9; reverse,5′-CGGTCGAAGTTTTCAGCGTTTC-3′, Sequence ID No. 10. The primer pair forPKC βII was (419 bp): forward, 5′-TATCTGGGATGGGGTGACAACC-3′, Sequence IDNo. 11; reverse, 5′-CGGTCGAAGTTTTCAGCGTTTC-3′, Sequence ID NO. 12. PCRproducts were separated on a 4% agarose gel and stained with ethidiumbromide. A representative gel is shown in FIG. 3. Gels were scanned andband intensities quantitated with BioImage software version 2.1 (AnnArbor, Mich.). The relative amount of sample mRNA was calculated bydividing the sample band intensity by the internal standard bandintensity. Specific amplification of mRNA was monitored by running PCRnegative controls consisting of tubes containing either sample RNAwithout reverse transcription, reverse transcribed sample without mimic,or mimic only. To ensure reproducibility of results, selected sampleswere amplified in duplicate. In addition, the fidelity of all PCRreactions was confirmed by DNA sequencing (Jiang et al., 1996).

[0117] Statistical Analysis:

[0118] Data were analyzed to determine the effects of carcinogen andpresence of tumor using one-way ANOVA. When P-values were <0.05 for theeffects of tumor or carcinogen, total means were separated usingDuncan's multiple range test.

Results

[0119] Colon Carcinoma Incidence:

[0120] There was no evidence of carcinoma in any saline injected animal,whereas 64% of carcinogen injected rats had carcinomas at the time ofdeath.

[0121] Effect of Carcinogen and Presence of Tumor on Fecal and MucosalPKC Isozyme mRNA Levels:

[0122] To determine the specificity of this non-invasive procedure, PKCβI and γ primers were used as negative controls (Davidson et al., 1994;Davidson et al., 1995). No amplified products were detected after 40cycles in any fecal poly A+ or scraped colonic mucosa total RNA samples(FIG. 3, lanes 3, 4, 6 and 7). However, both isozymes were detectedusing brain total RNA (positive control, lanes 2 and 5).

[0123] PCR products for PKC βII were detected in all fecal and mucosalsamples. Samples processed without reverse transcriptase were used asnegative controls and yielded no detectable amplified products (data notshown). Using semiquantitative mimic PCR, it was determined that fecalPKC βII mRNA levels were altered by the presence of a tumor withtumor-bearing animals having 3-fold higher (P<0.05) PKC βII expressionas compared with animals without tumors, as seen in FIG. 4A. Incontrast, there was no effect of tumor incidence on mucosal PKC βIIexpression. However, there was a significant effect (P<0.05) ofinjection on mucosal PKC βII expression. Specifically, carcinogen (AOM)injection increased mucosal PKC βII mRNA expression compared with salinecontrols (FIG. 4B).

[0124] Colonic mucosal PKC ζ expression in rats injected with AOM wasless than half (P<0.05) that of saline control, as shown in FIG. 5.Since tumor incidence exerts a reciprocal effect on fecal PKC ζ and PKCβII expression, data were also expressed as the ratio between PKC βIIand PKC ζ. The isozyme ratio was strongly related to tumor incidence,i.e. ratio for animals with tumors was 2.18±1.25 (n=9), animals withouttumors was 0.50±0.6 (n=26), P=0.025 (FIG. 6). These data demonstratethat PKC βII and PKC ζ may serve as non-invasive markers for developmentof colon tumors.

EXPERIMENT 5 Enhancement of Noninvasive mRNA-Gene Expression ProfilingUsing Biochip Technology

[0125] mRNA isolated from feces can be utilized in combination withcomplimentary DNA (cDNA) and oligonucleotide microarray technology inorder to noninvasively determine complex patterns of gene expression,and mutations (Bowtell, 1999; Duggan, 1999; Lipshutz, 1999). Biochiptechnology is described in many publications (including Bowtell, 1999;Duggan, 1999; Lipshutz, 1999 which are incorporated herein byreference), and is known in the art. This technology allows forlarge-scale, high-throughput monitoring of gene expression patterns ofup to 40,000 genes (Bowtell, 1999; Duggan, 1999; Lipshutz, 1999).Generated data provide insight into the extent of expression differencesunderlying colonic disease, e.g., malignancy, and reveal genes that mayprove useful as diagnostic or prognostic markers.

[0126] Description of the method:

[0127] 0.1-1 μg of fecal poly A+ RNA isolated from animal/human subjectsas previously described, is processed in strict accordance to thefollowing protocols or others known in the art. For example, followingfecal mRNA isolation, CDNA synthesis will be performed using selectprimers, such as, for example (a T7-(dT)₂₄-3′ primer:5′-GGCCAGTGAATTGTAATACGACTCACTAT-AGGGAGGCGG-(dT)₂₄-3′) (Sequence ID No.13). Subsequently, in vitro transcription is performed to generatelabeled samples for hybridization. This technology is known in the art.cRNA fragmentation, target hybridization, fluidics station setup, probearray washing and staining, probe array scan, and initial data analysisare performed according to procedures known in the art. The precisecomposition of the probe microarray can vary depending on the specificpackage of genes being surveyed. The microarrays are currently capableof simultaneously quantitating mRNA levels (gene expression) forthousands of genes in a single experiment. Quantitative changes in mRNAexpression patterns of approximately 2-fold or greater can be detected(Bowtell, 1999; Duggan, 1999; Lipshutz, 1999). With regard tospecificity, hybridization discrimination of low abundance transcriptsis currently 1:50,000-1:100,000.

[0128] Fecal (exfoliated colonic cell) mRNA isolation methodology incombination with Biochip technology can be utilized to assay for anumber of gene expression applications. For example:

[0129] 1. Tissue comparison: diseased (e.g., colon cancer, colitis) vs.unaffected colon, as a means of predicting disease onset.

[0130] 2. Time point experiments: determine patient status over time.

[0131] 3. Drug response in the body.

Explanation of GeneChip Probe Arrays

[0132] GeneChip probe arrays are known in the art and in essence aremanufactured using technology that combines photolithographic methodsand combinational chemistry. Tens to hundreds of thousands of differentoligonucleotide probes are synthesized, for example, in a 1.28 cm×1.28cm area on each array. Each probe type is located in a specific area onthe probe array called a probe cell. Each probe cell contains millionsof copies of a given probe. In use, biotin-labeled RNA fragments,referred to as the RNA targets, are hybridized to the probe array. Thehybridized probe array is stained with, for example, streptavidinphycoerythrin conjugate and scanned by the Hewlett-Packard (HP)GeneArray™ Scanner at the excitation wavelength of 488 nm. The amount oflight emitted at 570 nm is proportional to bound target at each locationon the probe array.

[0133] Target Preparation

[0134] Double stranded cDNA is synthesized from poly A+ messenger RNAisolated from tissue or cells. An in vitro reaction is then performed toproduce biotin-labeled cRNA from the cDNA. The cRNA is fragmented beforehybridization.

[0135] Target Hybridization

[0136] After the biotin-labeled cRNA is fragmented, a hybridizationcocktail is prepared, which includes the fragmented cRNA, probe arraycontrols, BSA, and herring sperm DNA. A cleanup procedure is performedon the hybridization cocktail after which approximately 200 μL isapplied to the probe array. It is then hybridized to the oligonucleotideprobes on the probe array during a 16-hour incubation at 45° C.

[0137] Probe Array Washing and Staining

[0138] Immediately following the hybridization, the hybridized probearray undergoes a washing and staining protocol as known in the art.

[0139] Probe Array Scan

[0140] Once the probe array has been hybridized, stained, and washed, itis scanned as known in the art.

[0141] Data Analysis

[0142] Data are analyzed using the GeneChip software available in theart. The data image is analyzed for probe intensities and results arereported in tabular and graphical formats.

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[0185] One skilled in the art readily appreciates that the presentinvention is well adapted to carry out the objectives and obtain theends and advantages mentioned as well as those inherent therein.Systems, biochemical compositions, treatments, methods, procedures andtechniques described herein are presently representative of thepreferred embodiments and are intended to be exemplary and are notintended as limitations of the scope. Changes therein and other useswill occur to those skilled in the art which are encompassed within thespirit of the invention or defined by the scope of the pending claims.

1 12 1 21 DNA RATTUS SP 1 ttgatgggga agatgaggag g 21 2 22 DNA RATTUS SP2 gaaatcagct tggtcgatgc tg 22 3 24 DNA RATTUS SP 3 tgtgatggag tatgtgaacggggg 24 4 24 DNA RATTUS SP 4 tcgaagttgg aggtgtctcg cttg 24 5 23 DNARATTUS SP 5 cgatggggtg gatgggatca aaa 23 6 35 DNA RATTUS SP 6 gtattcatgtcagggttgtc tggatttcgg gggcg 35 7 23 DNA RATTUS SP 7 cgatggggtggatgggatca aaa 23 8 22 DNA RATTUS SP 8 gtattcatgt cagggttgtc tg 22 9 36DNA RATTUS SP 9 tatctgggat ggggtgacaa ccgagatcat tgctta 36 10 22 DNARATTUS SP 10 cggtcgaagt tttcagcgtt tc 22 11 22 DNA RATTUS SP 11tatctgggat ggggtgacaa cc 22 12 22 DNA RATTUS SP 12 cggtcgaagt tttcagcgtttc 22

1. A method for non-invasively determining the expression of PKCisozymes in colonocytes of a patient comprising: directly isolating froma patient poly A+ RNA from feces, containing sloughed colonocytes; andassaying the isolated poly A+ RNA and determining the level, in theisolated A+ RNA, of mRNA encoding at least one PKC isozyme.
 2. Themethod of claim 1 , wherein the RNA is assayed using semi-quantitativeRT-PCR.
 3. The method of claim 1 , wherein the RNA is assayed usingbiochip microarray technology.
 4. The method of claim 1 , wherein atleast one PKC isozyme is selected from the group consisting of PKC ζ,PKC βII, PKC γ, and PKC βI.
 5. The method of claim 1 , wherein the levelof expression of PKC isozymes PKC ζ, and PKC βII is determined.
 6. Themethod of claim 5 , wherein the ratio of expression of PKC βII to PKC ζis determined.
 7. The method of claim 6 , further comprising the step ofcomparing the ratio of expression of PKC βII to PKC ζ in said patientwith similarly determined ratios of PKC βII to PKC ζ in other patientswith known conditions.
 8. The method of claim 7 , wherein the level ofexpression of PKC βII to PKC ζ in said patient is compared withsimilarly determined ratios of PKC βII to PKC ζ in at least two otherpatients, one with colon cancer and one without colon cancer.
 9. Themethod of claim 6 , wherein the level of PKC ζ is determined using theprimer pair having Sequence ID Numbers 7 and 8, and the level of PKC βIIis determined using the primer pair selected from the group consistingof the primers having Sequence ID Numbers 11, and
 12. 10. The method ofclaim 1 , wherein the poly A+ RNA is isolated from rectal eluateobtained at the time of a colonoscopy.
 11. A method for non-invasivelydetecting colonic biomarkers in a patient using fecal messenger RNAcomprising: directly isolating, from said patient, poly A+ RNA fromfeces containing sloughed colonocytes; and assaying the isolated poly A+RNA and determining the level, in the isolated poly A+ RNA, of mRNAencoding at least one colonic biomarker.
 12. The method of claim 11 ,wherein the RNA is assayed using semi-quantitative RT-PCR.
 13. Themethod of claim 11 , wherein the RNA is assayed using biochiptechnology.
 14. The method of claim 11 , wherein said at least onecolonic biomarker is a specific isozyme of PKC.
 15. The method of claim14 , wherein the specific isozyme of PKC is selected from the groupconsisting of PKC ζ, PKC βII, PKC γ, and PKC βI.
 16. The method of claim15 , wherein the level of expression of PKC isozymes PKC ζ and PKC βIIis determined.
 17. The method of claim 16 , wherein the ratio ofexpression of PKC βII to PKC ζ is determined.
 18. The method of claim 17, further comprising the step of comparing the ratio of expression ofPKC βII to PKC ζ in said patient with similarly determined ratios of PKCβII to PKC ζ in at least two other patients, one with colon cancer andone without colon cancer.
 19. The method of claim 16 , wherein the levelof PKC ζ is determined using at least one primer selected from the groupconsisting of the primers having Sequence ID Numbers 5, 6, 7, and 8, andthe level of PKC βII is determined using at least one primer selectedfrom the group consisting of the primers having Sequence ID Numbers 9,10, 11, and
 12. 20. The method of claim 11 , wherein the poly A+ RNA isisolated from rectal eluate obtained at the time of a colonoscopy.
 21. Amethod for non-invasively screening for colon cancer in a patientcomprising: detecting the expression of at least one specific biomarkerin sloughed coloncytes in said patient's feces; and correlating theexpression of said at least one specific biomarker with the presence orabsence of colon cancer in said patient.
 22. The method of claim 21 ,wherein said at least one specific biomarker is an isozyme selected fromthe group consisting of PKC ζ, PKC βII, PKC γ, and PKC βI.
 23. Themethod of claim 22 , wherein the level of expression of PKC isozymes PKCζ and PKC βII is determined.
 24. The method of claim 23 , wherein theratio of expression of PKC βII to PKC ζ is determined.
 25. The method ofclaim 21 , further comprising the step of comparing the ratio ofexpression of PKC βII to PKC ζ in said patient with similarly determinedratios of PKC βII to PKC ζ in at least two other patients, one withcolon cancer and one without colon cancer.
 26. The method of claim 23 ,wherein the level of PKC ζ is determined using at least one primerselected from the group consisting of the primers having Sequence IDNumbers 5, 6, 7, and 8, and the level of PKC βII is determined using atleast one primer selected from the group consisting of the primershaving Sequence ID Numbers 9, 10, 11, and
 12. 27. A method for isolatingpoly A+ RNA from feces comprising the steps of: homogenizing feces;adding a dilution buffer to the feces to form a fecal homogenate;centrifuging the fecal homogenate to create a supernatant; adding oligodT cellulose to the supernatant to form an oligo dT cellulosesupernatant mixture; mixing the oligo dT cellulose supernatant mixture;centrifuging the oligo dT cellulose supernatant mixture to form apellet; resuspending the pelleted resin with a binding buffer;centrifuging the resuspended resin to form a pellet and discarding thesupernatant; resuspending the pelleted resin with a wash buffer;centrifuging the resuspended resin to form a pellet and discarding thesupernatant; resuspending the pelleted resin with an elution buffer;eluting the poly A+ RNA by centrifugation; precipitating the poly A+ RNAby adding a precipitation solution and chilling the resuspension; andcentrifuging the chilled resuspension to pellet poly A+ RNA.
 28. Themethod of claim 27 wherein the amount of oligo dT cellulose added to thesupernatant is in an amount equal to 10% of the starting fecal weight.29. The method of claim 27 wherein the steps of centrifuging the oligodT cellulose supernatant mixture to form a pellet, and resuspending thepelleted resin with a binding buffer are performed more than once. 30.The method of claim 27 wherein the steps of centrifuging the resuspendedresin to form a pellet and discarding the supernatant, and resuspendingthe pelleted resin with a wash buffer are performed more than once. 31.The method of claim 27 wherein the elution buffer is about 65° C. 32.The method of claim 27 wherein the precipitation solution comprisesabout 60 μl 5M ammonium acetate, about 10 μg glycogen and about 2.5 vol100% ethanol.
 33. The method of claim 27 wherein the pelleted poly A+RNA is further resuspended in a water/EDTA solution.